Cystathionine-γ-lyase drives antioxidant defense in cysteine-restricted IDH1-mutant astrocytomas.

BACKGROUND: Mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) define glioma subtypes and are considered primary events in gliomagenesis, impacting tumor epigenetics and metabolism. IDH enzyme activity is crucial for the generation of reducing potential in normal cells, yet the impact of the mutation on the cellular antioxidant system in glioma is not understood. The aim of this study was to determine how glutathione (GSH), the main antioxidant in the brain, is maintained in IDH1-mutant gliomas, despite an altered NADPH/NADP balance. METHODS: Proteomics, metabolomics, metabolic tracer studies, genetic silencing, and drug targeting approaches in vitro and in vivo were applied. Analyses were done in clinical specimen of different glioma subtypes, in glioma patient-derived cell lines carrying the endogenous IDH1 mutation and corresponding orthotopic xenografts in mice. RESULTS: We find that cystathionine-γ-lyase (CSE), the enzyme responsible for cysteine production upstream of GSH biosynthesis, is specifically upregulated in IDH1-mutant astrocytomas. CSE inhibition sensitized these cells to cysteine depletion, an effect not observed in IDH1 wild-type gliomas. This correlated with an increase in reactive oxygen species and reduced GSH synthesis. Propargylglycine (PAG), a brain-penetrant drug specifically targeting CSE, led to delayed tumor growth in mice. CONCLUSIONS: We show that IDH1-mutant astrocytic gliomas critically rely on NADPH-independent de novo GSH synthesis via CSE to maintain the antioxidant defense, which highlights a novel metabolic vulnerability that may be therapeutically exploited.

Altered metabolic landscape in IDH-mutant gliomas affects phospholipid, energy, and oxidative stress pathways.

Heterozygous mutations in NADP-dependent isocitrate dehydrogenases (IDH) define the large majority of diffuse gliomas and are associated with hypermethylation of DNA and chromatin. The metabolic dysregulations imposed by these mutations, whether dependent or not on the oncometabolite D-2-hydroxyglutarate (D2HG), are less well understood. Here, we applied mass spectrometry imaging on intracranial patient-derived xenografts of IDH-mutant versus IDH wild-type glioma to profile the distribution of metabolites at high anatomical resolution in situ This approach was complemented by in vivo tracing of labeled nutrients followed by liquid chromatography-mass spectrometry (LC-MS) analysis. Selected metabolites were verified on clinical specimen. Our data identify remarkable differences in the phospholipid composition of gliomas harboring the IDH1 mutation. Moreover, we show that these tumors are characterized by reduced glucose turnover and a lower energy potential, correlating with their reduced aggressivity. Despite these differences, our data also show that D2HG overproduction does not result in a global aberration of the central carbon metabolism, indicating strong adaptive mechanisms at hand. Intriguingly, D2HG shows no quantitatively important glucose-derived label in IDH-mutant tumors, which suggests that the synthesis of this oncometabolite may rely on alternative carbon sources. Despite a reduction in NADPH, glutathione levels are maintained. We found that genes coding for key enzymes in de novo glutathione synthesis are highly expressed in IDH-mutant gliomas and the expression of cystathionine-ß-synthase (CBS) correlates with patient survival in the oligodendroglial subtype. This study provides a detailed and clinically relevant insight into the in vivo metabolism of IDH1-mutant gliomas and points to novel metabolic vulnerabilities in these tumors.

Targeted Proteomics to Assess the Response to Anti-Angiogenic Treatment in Human Glioblastoma (GBM).

Glioblastoma (GBM) is a highly aggressive primary brain tumor with dismal outcome for affected patients. Because of the significant neo-angiogenesis exhibited by GBMs, anti-angiogenic therapies have been intensively evaluated during the past years. Recent clinical studies were however disappointing, although a subpopulation of patients may benefit from such treatment. We have previously shown that anti-angiogenic targeting in GBM increases hypoxia and leads to a metabolic adaptation toward glycolysis, suggesting that combination treatments also targeting the glycolytic phenotype may be effective in GBM patients. The aim of this study was to identify marker proteins that are altered by treatment and may serve as a short term readout of anti-angiogenic therapy. Ultimately such proteins could be tested as markers of efficacy able to identify patient subpopulations responsive to the treatment. We applied a proteomics approach based on selected reaction monitoring (SRM) to precisely quantify targeted protein candidates, selected from pathways related to metabolism, apoptosis and angiogenesis. The workflow was developed in the context of patient-derived intracranial GBM xenografts developed in rodents and ensured the specific identification of human tumor versus rodent stroma-derived proteins. Quality control experiments were applied to assess sample heterogeneity and reproducibility of SRM assays at different levels. The data demonstrate that tumor specific proteins can be precisely quantified within complex biological samples, reliably identifying small concentration differences induced by the treatment. In line with previous work, we identified decreased levels of TCA cycle enzymes, including isocitrate dehydrogenase, whereas malectin, calnexin, and lactate dehydrogenase A were augmented after treatment. We propose the most responsive proteins of our subset as potential novel biomarkers to assess treatment response after anti-angiogenic therapy that warrant future analysis in clinical GBM samples.

Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma.

L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes.

Bevacizumab treatment induces metabolic adaptation toward anaerobic metabolism in glioblastomas.

Anti-angiogenic therapy in glioblastoma (GBM) has unfortunately not led to the anticipated improvement in patient prognosis. We here describe how human GBM adapts to bevacizumab treatment at the metabolic level. By performing (13)C6-glucose metabolic flux analysis, we show for the first time that the tumors undergo metabolic re-programming toward anaerobic metabolism, thereby uncoupling glycolysis from oxidative phosphorylation. Following treatment, an increased influx of (13)C6-glucose was observed into the tumors, concomitant to increased lactate levels and a reduction of metabolites associated with the tricarboxylic acid cycle. This was confirmed by increased expression of glycolytic enzymes including pyruvate dehydrogenase kinase in the treated tumors. Interestingly, L-glutamine levels were also reduced. These results were further confirmed by the assessment of in vivo metabolic data obtained by magnetic resonance spectroscopy and positron emission tomography. Moreover, bevacizumab led to a depletion in glutathione levels indicating that the treatment caused oxidative stress in the tumors. Confirming the metabolic flux results, immunohistochemical analysis showed an up-regulation of lactate dehydrogenase in the bevacizumab-treated tumor core as well as in single tumor cells infiltrating the brain, which may explain the increased invasion observed after bevacizumab treatment. These observations were further validated in a panel of eight human GBM patients in which paired biopsy samples were obtained before and after bevacizumab treatment. Importantly, we show that the GBM adaptation to bevacizumab therapy is not mediated by clonal selection mechanisms, but represents an adaptive response to therapy.

Colorectal cancer derived organotypic spheroids maintain essential tissue characteristics but adapt their metabolism in culture.

BACKGROUND: Organotypic tumor spheroids, a 3D in vitro model derived from patient tumor material, preserve tissue heterogeneity and retain structural tissue elements, thus replicating the in vivo tumor more closely than commonly used 2D and 3D cell line models. Such structures harbour tumorigenic cells, as revealed by xenograft implantation studies in animal models and maintain the genetic makeup of the original tumor material. The aim of our work was a morphological and proteomic characterization of organotypic spheroids derived from colorectal cancer tissue in order to get insight into their composition and associated biology. RESULTS: Morphological analysis showed that spheroids were of about 250 µm in size and varied in structure, while the spheroid cells differed in shape and size and were tightly packed together by desmosomes and tight junctions. Our proteomic data revealed significant alterations in protein expression in organotypic tumor spheroids cultured as primary explants compared to primary colorectal cancer tissue. Components underlying cellular and tissue architecture were changed; nuclear DNA/ chromatin maintenance systems were up-regulated, whereas various mitochondrial components were down-regulated in spheroids. Most interestingly, the mesenchymal cells appear to be substantial component in such cellular assemblies. Thus the observed changes may partly occur in this cellular compartment. Finally, in the proteomics analysis stem cell-like characteristics were observed within the spheroid cellular assembly, reflected by accumulation of Alcam, Ctnnb1, Aldh1, Gpx2, and CD166. These findings were underlined by IHC analysis of Ctnnb1, CD24 and CD44, therefore warranting closer investigation of the tumorigenic compartment in this 3D culture model for tumor tissue. CONCLUSIONS: Our analysis of organotypic CRC tumor spheroids has identified biological processes associated with a mixture of cell types and states, including protein markers for mesenchymal and stem-like cells. This 3D tumor model in which tumor heterogeneity is preserved may represent an advantageous model system to investigate novel therapeutic approaches.

Increased mitochondrial activity in a novel IDH1-R132H mutant human oligodendroglioma xenograft model: in situ detection of 2-HG and α-KG.

BACKGROUND: Point mutations in genes encoding NADP+-dependent isocitrate dehydrogenases (especially IDH1) are common in lower grade diffuse gliomas and secondary glioblastomas and occur early during tumor development. The contribution of these mutations to gliomagenesis is not completely understood and research is hampered by the lack of relevant tumor models. We previously described the development of the patient-derived high-grade oligodendroglioma xenograft model E478 that carries the commonly occurring IDH1-R132H mutation. We here report on the analyses of E478 xenografts at the genetic, histologic and metabolic level. RESULTS: LC-MS and in situ mass spectrometric imaging by LESA-nano ESI-FTICR revealed high levels of the proposed oncometabolite D-2-hydroxyglutarate (D-2HG), the product of enzymatic conversion of α-ketoglutarate (α-KG) by IDH1-R132H, in the tumor but not in surrounding brain parenchyma. α-KG levels and total NADP+-dependent IDH activity were similar in IDH1-mutant and -wildtype xenografts, demonstrating that IDH1-mutated cancer cells maintain α-KG levels. Interestingly, IDH1-mutant tumor cells in vivo present with high densities of mitochondria and increased levels of mitochondrial activity as compared to IDH1-wildtype xenografts. It is not yet clear whether this altered mitochondrial activity is a driver or a consequence of tumorigenesis. CONCLUSIONS: The oligodendroglioma model presented here is a valuable model for further functional elucidation of the effects of IDH1 mutations on tumor metabolism and may aid in the rational development of novel therapeutic strategies for the large subgroup of gliomas carrying IDH1 mutations.

The soluble form of the tumor suppressor Lrig1 potently inhibits in vivo glioma growth irrespective of EGF receptor status.

BACKGROUND: Deregulated growth factor signaling is a major driving force in the initiation and progression of glioblastoma. The tumor suppressor and stem cell marker Lrig1 is a negative regulator of the epidermal growth factor receptor (EGFR) family. Here, we addressed the therapeutic potential of the soluble form of Lrig1 (sLrig1) in glioblastoma treatment and the mechanism of sLrig1-induced growth inhibition. METHODS: With use of encapsulated cells, recombinant sLrig1 was locally delivered in orthotopic glioblastoma xenografts generated from freshly isolated patient tumors. Tumor growth and mouse survival were evaluated. The efficacy of sLrig1 and the affected downstream signaling was studied in vitro and in vivo in glioma cells displaying variable expression of wild-type and/or a constitutively active EGFR mutant (EGFRvIII). RESULTS: Continuous interstitial delivery of sLrig1 in genetically diverse patient-derived glioma xenografts led to strong tumor growth inhibition. Glioma cell proliferation in vitro and tumor growth in vivo were potently inhibited by sLrig1, irrespective of EGFR expression levels. Of importance, tumor growth was also suppressed in EGFRvIII-driven glioma. sLrig1 induced cell cycle arrest without changing total receptor level or phosphorylation. Affected downstream effectors included MAP kinase but not AKT signaling. Of importance, local delivery of sLrig1 into established tumors led to a 32% survival advantage in treated mice. CONCLUSIONS: To our knowledge, this is the first report demonstrating that sLrig1 is a potent inhibitor of glioblastoma growth in clinically relevant experimental glioma models and that this effect is largely independent of EGFR status. The potent anti-tumor effect of sLrig1, in combination with cell encapsulation technology for in situ delivery, holds promise for future treatment of glioblastoma.

Combined effect of tissue stabilization and protein extraction methods on phosphoprotein analysis.

Preanalytical conditions applied during sample collection and processing can affect the detection or quantification of unstable phosphoprotein biomarkers. We evaluated the consequences of tissue stabilization and protein extraction methods on phosphoprotein analysis. The effects of stabilization techniques (heat stabilization, snap-freezing) and time on the levels of phosphoproteins, including phospho-Akt, p-ERK 1/2, p-IkBα, p-JNK, and p38 MAPK, were evaluated using a BioPlex phosphoprotein assay. Additionally, two different protein extraction protocols, using different extraction buffers (8 M urea buffer, or Bio-Rad buffer without urea) were tested. For snap-frozen samples, protein extraction yields were comparable with the two buffer systems. For heat-stabilized samples, total protein yields were significantly lower following extraction in non-urea buffer. However, the concentrations of specific phosphoproteins were significantly higher in heat-stabilized samples than in the corresponding snap-frozen samples, indicating that this tissue processing method better preserved phosphoproteins. Significant differences were found between the measured phosphoprotein levels in heat-stabilized and snap-frozen tissue, suggesting that alterations occur very rapidly after tissue excision. Our results suggest that heat stabilization can be used as a tissue processing method for subsequent phosphoprotein analyses, but also suggest that the BioPlex phosphoprotein assay could be used as a possible quality control method to assess tissue sample integrity.

Identification of differentially expressed proteins in curcumin-treated prostate cancer cell lines.

Due to high prevalence and slow progression of prostate cancer, primary prevention appears to be attractive strategy for its eradication. During the last decade, curcumin (diferuloylmethane), a natural compound from the root of turmeric (Curcuma longa), was described as a potent chemopreventive agent. Curcumin exhibits anti-inflammatory, anticarcinogenic, antiproliferative, antiangiogenic, and antioxidant properties in various cancer cell models. This study was designed to identify proteins involved in the anticancer activity of curcumin in androgen-dependent (22Rv1) and -independent (PC-3) human prostate cancer cell lines using two-dimensional difference in gel electrophoresis (2D-DIGE). Out of 425 differentially expressed spots, we describe here the MALDI-TOF-MS analysis of 192 spots of interest, selected by their expression profile. This approach allowed the identification of 60 differentially expressed proteins (32 in 22Rv1 cells and 47 in PC-3 cells). Nineteen proteins are regulated in both cell lines. Further bioinformatic analysis shows that proteins modulated by curcumin are implicated in protein folding (such as heat-shock protein PPP2R1A; RNA splicing proteins RBM17, DDX39; cell death proteins HMGB1 and NPM1; proteins involved in androgen receptor signaling, NPM1 and FKBP4/FKBP52), and that this compound could have an impact on miR-141, miR-152, and miR-183 expression. Taken together, these data support the hypothesis that curcumin is an interesting chemopreventive agent as it modulates the expression of proteins that potentially contribute to prostate carcinogenesis.

Atrazine and PCB 153 and their effects on the proteome of subcellular fractions of human MCF-7 cells.

Several man-made organic pollutants including polychlorinated biphenyls (PCBs) and several pesticides may exhibit endocrine disrupting (ED) properties. These ED molecules can be comparatively persistent in the environment, and have shown to perturb hormonal activity and several physiological functions. The objective of this investigation was to study the impact of PCB 153 and atrazine on human MCF-7 cells, and to search for marker proteins of their exposure. Cells were exposed to environmentally high but relevant concentrations of atrazine (200ppb), PCB 153 (500ppb), 17-ß estradiol (positive control, 10nM) and DMSO (0.1%, negative control) for t=36h (n=3 replicates/exposure group). Proteins from cell membrane and cytosol were isolated, and studied by 2D-DiGE. Differentially regulated proteins were trypsin-digested and identified by MALDI-ToF-ToF and NCBInr database. A total of 36 differentially regulated proteins (>|1.5| fold change, P<0.05) were identified in the membrane fraction and 22 in the cytosol, and were mainly involved in cell structure and in stress response, but also in xenobiotic metabolism. 67% (membrane) and 50% (cytosol) of differentially regulated proteins were more abundant following atrazine exposure whereas nearly 100% (membrane) and 45% (cytosol) were less abundant following PCB 153 exposure. Western blots of selected proteins (HSBP1, FKBP4, STMN1) confirmed 2D-DiGE results. This study emphasizes the numerous potential effects that ED compounds could have on exposed humans.

Anti-VEGF treatment reduces blood supply and increases tumor cell invasion in glioblastoma.

Bevacizumab, an antibody against vascular endothelial growth factor (VEGF), is a promising, yet controversial, drug in human glioblastoma treatment (GBM). Its effects on tumor burden, recurrence, and vascular physiology are unclear. We therefore determined the tumor response to bevacizumab at the phenotypic, physiological, and molecular level in a clinically relevant intracranial GBM xenograft model derived from patient tumor spheroids. Using anatomical and physiological magnetic resonance imaging (MRI), we show that bevacizumab causes a strong decrease in contrast enhancement while having only a marginal effect on tumor growth. Interestingly, dynamic contrast-enhanced MRI revealed a significant reduction of the vascular supply, as evidenced by a decrease in intratumoral blood flow and volume and, at the morphological level, by a strong reduction of large- and medium-sized blood vessels. Electron microscopy revealed fewer mitochondria in the treated tumor cells. Importantly, this was accompanied by a 68% increase in infiltrating tumor cells in the brain parenchyma. At the molecular level we observed an increase in lactate and alanine metabolites, together with an induction of hypoxia-inducible factor 1α and an activation of the phosphatidyl-inositol-3-kinase pathway. These data strongly suggest that vascular remodeling induced by anti-VEGF treatment leads to a more hypoxic tumor microenvironment. This favors a metabolic change in the tumor cells toward glycolysis, which leads to enhanced tumor cell invasion into the normal brain. The present work underlines the need to combine anti-angiogenic treatment in GBMs with drugs targeting specific signaling or metabolic pathways linked to the glycolytic phenotype.

Cortisol is a potent modulator of lipopolysaccharide-induced interferon signaling in macrophages.

The effects of cortisol (CORT) on resting and lipopolysaccharide (LPS)-activated monocyte-derived THP-1 macrophages were investigated by proteomics. Forty-seven proteins were found to be modulated, 20 by CORT, 11 by LPS, and 16 by CORT and LPS. Cortisol-sensitive chaperones and cytoskeletal proteins were mostly repressed. HCLS1, MGN, and MX1 were new proteins identified to be under the transcriptional control of this steroid and new CORT-sensitive variants of MX1, SYWC and IFIT3 were found. FKBP51, a known CORT target gene, showed the strongest response to CORT and synergism with LPS. In resting THP-1 macrophages, 18 proteins were modulated by CORT, with 15 being down-regulated. Activation of macrophages by LPS was associated with enhanced expression of immune response and metabolic proteins. In activated macrophages, CORT had a more equilibrated effect and almost all metabolism-related proteins were up-regulated, whereas immune response proteins were mostly down-regulated. The majority of the LPS up-regulated immune response-related proteins are known interferon (IFN) target genes (IFIT3, MX1, SYWC, PSME2) suggesting activation of the IRF3 signaling pathway. They were all suppressed by CORT. This is the first proteomics study to investigate the effects of CORT on activated immune cells.

Extensive phosphorylation flanking the C-terminal functional domains of the measles virus nucleoprotein.

The measles virus nucleoprotein (vNP) is the first and most abundant protein in infected cells. It plays numerous important roles including the encapsidation of genomic viral RNA and the transcription of viral proteins. Intricate interactions with host cell proteins rely on the structural integrity of its functional domains. Although some of these functional domains are known, their structural features are still poorly understood. Here we identified multiple isoforms of measles vNP by two-dimensional differential gel electrophoresis (2D-DIGE) and 2D Western blot. These isoforms were further analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)/TOF using MS (PMF) and MSMS (PSD) and electrospray ionization (ESI)-ion trap using LC-ESI-ion trap MS(1), MS(2) (neutral loss), MS(3) (phosphosite). Both recombinant NP (rNP) and vNP were α-acetylated at the N-terminus. After tryptic or chymotryptic digestion, phosphopeptides were enriched and nine phosphorylation sites were identified and localized in the rNP, seven of which were also phosphorylated in vNP, probably by casein kinase 2. The phosphosites were all found within the intrinsically unstructured C-terminal domain. They clustered around functional domains involved in transcription and replication, as well as in sequences interacting with host-cell proteins. This underlines the importance of these post-translational modifications.

Glioma proteomics: status and perspectives.

High grade gliomas are the most common brain tumors in adults and their malignant nature makes them the fourth biggest cause of cancer death. Major efforts in neuro-oncology research are needed to reach similar progress in treatment efficacy as that achieved for other cancers in recent years. In addition to the urgent need to identify novel effective drug targets against malignant gliomas, the search for glioma biomarkers and grade specific protein signatures will provide a much needed contribution to diagnosis, prognosis, treatment decision and assessment of treatment response. Over the past years glioma proteomics has been attempted at different levels, including proteome analysis of patient biopsies and bodily fluids, of glioma cell lines and animal models. Here we provide an extensive review of the outcome of these studies in terms of protein identifications (protein numbers and regulated proteins), with an emphasis on the methods used and the limitations of the studies with regard to biomarker discovery. This is followed by a perspective on novel technologies and on the potential future contribution of proteomics in a broad sense to understanding glioma biology.

Effects of the endocrine disruptors atrazine and PCB 153 on the protein expression of MCF-7 human cells.

Polychlorinated biphenyls (PCBs) and a number of pesticides can act as endocrine disrupting compounds (EDCs). These molecules exhibit hormonal activity in vivo, and can therefore interact and perturb normal physiological functions. Many of these compounds are persistent in the environment, and their bioaccumulation may constitute a significant threat for human health. Physiological abnormalities following exposure to these xenobiotic compounds go along with alterations at the protein level of individual cells. In this study, MCF-7 cells were exposed to environmentally relevant concentrations of atrazine, PCB153 (100 ppb, respectively), 17-beta estradiol (positive control, 10 nM) and a negative control (solvent) for t = 24 h (n = 3 replicates/exposure group). After trizol extraction and protein solubilization, protein expression levels were studied by 2D-DIGE. Proteins differentially expressed were excised, trypsin-digested, and identified by MALDI-ToF-ToF, followed by NCBInr database search. 2D-DIGE experiments demonstrated that 49 spots corresponding to 29 proteins were significantly differentially expressed in MCF-7 cells (>1.5-fold, P < 0.05, Student's paired t test). These proteins belonged to various cellular compartments (nucleus, cytosol, membrane), and varied in function; 88% of proteins were down-regulated during atrazine exposure, whereas 75% of proteins were up-regulated by PCB153. Affected proteins included those regulating oxidative stress such as superoxide dismutase and structural proteins such as actin or tropomyosin, which may explain morphological changes of cells already observed under the microscope. This study highlights the susceptibility of human cells to compounds with endocrine disrupting properties.

Proteomic analysis of the cortisol-mediated stress response in THP-1 monocytes using DIGE technology.

The glucocorticoid (GC) cortisol, the main mediator of the hypothalamic-pituitary-adrenal axis has many implications in metabolism, stress response and the immune system. Its function is mediated via binding to the glucocorticoid receptor (GR), a member of the superfamily of ligand-activated nuclear hormone receptors. The activity of the ligated GR results from its binding as a transcription factor to glucocorticoid response elements (GREs). Two-dimensional gel electrophoresis with DIGE (fluorescence difference gel electrophoresis) technology was applied to study the effects of cortisol on the human THP-1 monocytic cell line. A total of 28 cortisol-modulated proteins were identified belonging to five functional groups: cytoskeleton (8), chaperones (9), immune response (4), metabolism (3) and transcription/translation (4). Their corresponding genes were screened for putative GREs in their + 10 kb/- 0.2 kb promoter regions including all alternative promoters available within the Database for Transcription Start Sites (DBTSS). FKBP51, known to be induced by cortisol, was identified as the strongest differentially expressed protein, and contains the highest number of strict GREs. Genomic analysis of five alternative FKBP5 promoter regions suggests GC inducibility of all transcripts. Additionally, proteomics (2D DIGE and 2D immunoblotting) revealed the existence of several FKBP51 isoforms, which were not previously described. To our knowledge this is the first proteomic study that addresses the effects of cortisol on immune cells. FKBP51 isoforms found on the gel map were linked to alternative promoter usage on the genetic level, successfully correlating both the specific proteomic and genomic findings.

Measles virus genotyping by nucleotide-specific multiplex PCR.

A simple genotyping method based on multiplex PCR has been developed to discriminate between all active measles virus (MV) clades and genotypes (A, B3.1, B3.2, C2, D2-D9, G2-G3, and H1-H2). The sequencing reaction was replaced by six multiplex PCRs: one to identify the clade and five to identify the respective genotype. Primers were sensitive to clade- and genotype-specific nucleotides and generated fragments of type-specific sizes that were analyzed by conventional agarose gel electrophoresis. On the basis of all published MV sequences, positive and negative predictive values of 99.2% and 98.6% were calculated. Variability in the primer binding sites, which could potentially reduce sensitivity, was very limited among published sequences. As new genotypes are described, additional specific primers can be included in the multiplex PCR with relative ease. Although sequencing remains the "gold standard," the present method should facilitate MV genotyping especially in developing countries and will therefore contribute to enhanced MV control and elimination strategies as recommended by the World Health Organization.

Low genetic diversity despite hyperendemicity of hepatitis B virus genotype E throughout West Africa.

Sub-Saharan Africa suffers from an excessively high endemicity of hepatitis B virus (HBV), but little is known about the prevalent genotypes. In this study, we investigated the PreS1/PreS2/S genes of 127 viruses obtained from 12 locations in Mali, Burkina Faso, Togo, Benin, Nigeria, Cameroon, and the Democratic Republic of Congo. Except for those obtained from the Cameroon HIV cohort (18/22 HBV genotype A), 96 of 105 sequences belonged to HBV genotype E (HBV/E), and viral DNA was very similar (1.67% diversity) throughout this vast HBV/E crescent, which spans 6000 km across Africa. The low diversity suggests that HBV/E may have a short evolutionary history. Considering a typical mutation rate of DNA viruses, it would take only 200 years for the strain diversity of HBV/E viruses to develop from a single introductory event. The relatively recent introduction of HBV/E into humans would also explain its conspicuous absence in the Americas, despite the forced immigration of slaves from west Africa, until the early 19th century. Infection during infancy is mostly associated with chronic carrier status, and this combination can account for the explosive spread of virtually identical viruses within a community, but whether other routes of long-range transmissions must be considered becomes an important question.

Limited diversity of measles field isolates after a national immunization day in Burkina Faso: progress from endemic to epidemic transmission?

Despite recent National Immunization Days in Burkina Faso, the rural province of Houët reported >400 measles cases in 2001 (82% not vaccinated). Phylogenetic analysis of 58 measles virus field isolates plus the first sequences from the Democratic Republic of the Congo and the Republic of Congo are reported. All viruses were genotype B3, which is common in the region. In Houët, there were two geographically confined genetic variants, suggesting two independent importation events. Strain diversity in Houët (1.5%) and the Congos was limited in comparison with Ibadan, Nigeria (4.6%), where measles is endemic. Strain variability, assessed by heteroduplex mobility assay, confirmed these findings. Despite large local pools of susceptible persons even after several rounds of vaccination, the limited strain diversity suggests that parts of rural Burkina Faso may be moving from an endemic to an epidemic transmission pattern of measles virus.